A cancer protein microarray platform using antibody fragments and its clinical applications

Antibody microarrays have shown great potential for measurement of either a spectrum of target proteins in proteomics or disease-associated antigens in molecular diagnostics. Despite its importance, the applications of antibody microarrays are still limited by a variety of fundamental problems. Among them, cross-reactivity significantly limits the multiplexing ability in parallel sandwich immunoassays. As a result, it is very important to design new capture probes in order to incorporate a universal label into the assay configuration. In this report, an antibody fragments (F(ab')2) microarray platform for serum tumor markers was developed. Each antigen was detected at different concentrations to assemble its calibration curve, and combinations of different markers were tested to examine the specificity of simultaneous detection based on the F(ab')2 microarrays. Diagnostics of serum samples with this cancer antibody microarray platform and immunoradiometric assays (IRMA) were also performed. Wide range calibration curves (0-1280 U mL(-1)) were obtained for each tumor marker. Comparative studies demonstrated that such F(ab')2 microarrays exhibited both moderately improved sensitivity and better specificity than full-sized monoclonal antibody microarrays. It is also demonstrated that this microarray platform is quantitative, highly specific and reasonably sensitive. More importantly, clinical applications of our F(ab')2 microarray platform for upwards of 100 patient serum samples clearly show its potential in cancer diagnostics.     

Temperature scans/cycles for the detection of low abundant DNA point mutations on microarrays

The possibility to detect low abundant DNA point mutations is essential for early cancer diagnosis and/or prognosis. Furthermore, in order to be less invasive, the somatic mutations are not only sought in tumor extract samples but also from body fluids or stools rendering their content even more diluted compared to the wild type sequences. In this short communication, we propose two protocols based on temperature scans or cycles for the enrichment of the mutation strands hybridized on microarrays. We predict numerically and confirm experimentally a 10-fold increase in the fraction of mutated DNA hybridized on the microarray compared to the sample content. Coupled to more standard solution phase enrichment techniques, it would be possible to lower by one order of magnitude the current detection limit with the advantage of multiple mutation detections offered by the microarray technology.     

A Java-based tool for the design of classification microarrays

Background  

Classification microarrays are used for purposes such as identifying strains of bacteria and determining genetic relationships to understand the epidemiology of an infectious disease. For these cases, mixed microarrays, which are composed of DNA from more than one organism, are more effective than conventional microarrays composed of DNA from a single organism. Selection of probes is a key factor in designing successful mixed microarrays because redundant sequences are inefficient and limited representation of diversity can restrict application of the microarray. We have developed a Java-based software tool, called PLASMID, for use in selecting the minimum set of probe sequences needed to classify different groups of plasmids or bacteria.     

Restriction site tagged (RST) microarrays: a novel technique to study the species composition of complex microbial systems

We have developed a new type of microarray, restriction site tagged (RST), for example NotI, microarrays. In this approach only sequences surrounding specific restriction sites (i.e. NotI linking clones) were used for generating microarrays. DNA was labeled using a new procedure, NotI representation, where only sequences surrounding NotI sites were labeled. Due to these modifications, the sensitivity of RST microarrays increases several hundred-fold compared to that of ordinary genomic microarrays. In a pilot experiment we have produced NotI microarrays from Gram-positive and Gram-negative bacteria and have shown that even closely related Escherichia coli strains can be easily discriminated using this technique. For example, two E.coli strains, K12 and R2, differ by less than 0.1% in their 16S rRNA sequences and thus the 16S rRNA sequence would not easily discriminate between these strains. However, these strains showed distinctly different hybridization patterns with NotI microarrays. The same technique can be adapted to other restriction enzymes as well. This type of microarray opens the possibility not only for studies of the normal flora of the gut but also for any problem where quantitative and qualitative analysis of microbial (or large viral) genomes is needed.     

Electronic microarray analysis of 16S rDNA amplicons for bacterial detection

Electronic microarray technology is a potential alternative in bacterial detection and identification. However, conditions for bacterial detection by electronic microarray need optimization. Using the NanoChip electronic microarray, we investigated eight marine bacterial species. Based on the 16S rDNA sequences of these species, we constructed primers, reporter probes, and species-specific capture probes. We carried out two separate analyses for longer (533 bp) and shorter (350 and 200 bp) amplified products (amplicons). To detect simultaneously the hybridization signals for the 350- and 200-bp amplicons, we designed a common reporter probe from an overlapping sequence within both fragments. We developed methods to optimize detection of hybridization signals for processing the DNA chips. A matrix analysis was performed for different bacterial species and complementary capture probes on electronic microarrays. Results showed that, when using the longer amplicon, not all bacterial targets hybridized with the complementary capture probes, which was characterized by the presence of false-positive signals. However, with the shorter amplicons, all bacterial species were correctly and completely detected using the constructed complementary capture probes.     

Consensus PCR and microarray for diagnosis of the genus Staphylococcus, species, and methicillin resistance.

We propose the use of DNA microarray for the discrimination of homologous products after a single PCR amplification with consensus primers. The method was applied to Staphylococcus identification. The femA nucleotide sequences, which are phylogenetically conserved among the staphylococci, were first amplified using a consensus primer pair together with the mecA sequence, a molecular marker for methicillin resistance. Products were then identified on a glass array. The microarray contained five selective DNA capture probes for the simultaneous and differential identification of the five most clinically relevant staphylococcal species (S. aureus, S. epidermidis, S. haemolyticus, S. hominis, and S. saprophyticus), while a consensus capture probe could detect all femA sequences, allowing the identification of the genus Staphylococcus. The mecA sequence hybridized to a specific capture probe. The identification was univocal because only a single capture probe had to be present for each sequence to be identified. The hybridization and identification processes were completed in less than 2 h. Current results demonstrate that low-density microarrays are powerful multigenotypic post-PCR analyzers and could compete with conventional bacteria identification.     

Protein microarray on-demand: a novel protein microarray system

We describe a novel, simple and low-cost protein microarray strategy wherein the microarrays are generated by printing expression ready plasmid DNAs onto slides that can be converted into protein arrays on-demand. The printed expression plasmids serve dual purposes as they not only direct the synthesis of the protein of interest; they also serve to capture the newly synthesized proteins through a high affinity DNA-protein interaction. To accomplish this we have exploited the high-affinity binding (approximately 3-7 x 10 (-13) M) of E. coli Tus protein to Ter, a 20 bp DNA sequence involved in the regulation of E. coli DNA replication. In our system, each protein of interest is synthesized as a Tus fusion protein and each expression construct directing the protein synthesis contains embedded Ter DNA sequence. The embedded Ter sequence functions as a capture reagent for the newly synthesized Tus fusion protein. This "all DNA" microarray can be converted to a protein microarray on-demand without need for any additional capture reagent.     

Array-Based Approaches for the Identification of Epigenetic Silenced Tumor Suppressor Genes

Carcinogenesis involves the inactivation or inhibition of genes that function as tumor suppressors. Deletions, mutations, or epigenetic silencing of tumor suppressor genes can lead to altered growth, differentiation, and apoptosis. DNA methylation and histone modifications are important epigenetic mechanisms of gene regulation and play essential roles both independently and cooperatively in tumor initiation and progression. Realization that many tumor suppressor genes are silenced by epigenetic mechanisms has stimulated discovery of novel tumor suppressor genes. One of the most useful of these approaches is an epigenetic reactivation screening strategy that combines treatment of cancer cells in vitro with DNA methyltransferase and/or histone deacetylase (HDAC) inhibitors, followed by global gene expression analysis using microarrays, to identify upregulated genes. This approach is most effective when complemented by microarray analyses to identify genes repressed in primary tumors. Recently, using cancer cell lines treated with a DNA methylation inhibitor and/or a HDAC inhibitor in conjunction with cDNA microarray analysis, candidate tumor suppressor genes, which are subject to epigenetic silencing, have been identified in endometrial, colorectal, esophageal, and pancreatic cancers. An increasing number of studies have utilized epigenetic reactivation screening to discover novel tumor suppressor genes in cancer. The results of some of the most recent studies are highlighted in this review.     

Recent developments in DNA microarrays

DNA microarrays are used to quantify tens of thousands of DNA or RNA sequences in a single assay. Upon their introduction approximately six years ago, DNA microarrays were viewed as a disruptive technology that would fundamentally alter the scientific landscape. Supporting this view, the number of applications of DNA microarray technology has since expanded exponentially. Here, we review recent advances in microarray technology and selected new applications of the technology.     

High-throughput capture of nucleotide sequence polymorphisms in three Brassica species (Brassicaceae) using DNA microarrays

? Premise of the study: To capture molecular markers that are applicable to environmental risk assessment of genetically modified oilseed rape, and to streamline their development, we screened variations in nucleotide sequences of three Brassica species by DNA microarray analysis. ? Methods and Results: Using the Affymetrix GeneChip Arabidopsis ATH1 Genome Array, we monitored gene expression at 22810 loci among the Brassica species and picked out 192 putative polymorphic loci. We sequenced 25 of these and successfully aligned them among all three species. All 25 loci possessed some interspecific and at times intraspecific nucleotide variation. ? Conclusions: DNA microarray analysis effectively detected a large number of nucleotide sequence variations among closely related Brassica species. The polymorphic regions will allow the subsequent development of functional gene markers.     

Interpreting expression profiles of cancers by genome-wide survey of breadth of expression in normal tissues

A critical and difficult part of studying cancer with DNA microarrays is data interpretation. Besides the need for data analysis algorithms, integration of additional information about genes might be useful. We performed genome-wide expression profiling of 36 types of normal human tissues and identified 2503 tissue-specific genes. We then systematically studied the expression of these genes in cancers by reanalyzing a large collection of published DNA microarray datasets. We observed that the expression level of liver-specific genes in hepatocellular carcinoma (HCC) correlates with the clinically defined degree of tumor differentiation. Through unsupervised clustering of tissue-specific genes differentially expressed in tumors, we extracted expression patterns that are characteristic of individual cell types, uncovering differences in cell lineage among tumor subtypes. We were able to detect the expression signature of hepatocytes in HCC, neuron cells in medulloblastoma, glia cells in glioma, basal and luminal epithelial cells in breast tumors, and various cell types in lung cancer samples. We also demonstrated that tissue-specific expression signatures are useful in locating the origin of metastatic tumors. Our study shows that integration of each gene's breadth of expression (BOE) in normal tissues is important for biological interpretation of the expression profiles of cancers in terms of tumor differentiation, cell lineage, and metastasis.     

DNA microarrays with unimolecular hairpin double-stranded DNA probes: fabrication and exploration of sequence-specific DNA/protein interactions

We have fabricated double-stranded DNA (dsDNA) microarrays containing unimolecular hairpin dsDNA probes immobilized on glass slides. The unimolecular hairpin dsDNA microarrays were manufactured by four steps: Firstly, synthesizing single-stranded DNA (ssDNA) oligonucleotides with two reverse-complementary sequences at 3' hydroxyl end and an overhang sequence at 5' amino end. Secondly, microspotting ssDNA on glutaraldehyde-derived glass slide to form ssDNA microarrays. Thirdly, annealing two reverse-complementary sequences to form hairpin primer at 3' end of immobilized ssDNA and thus to create partial-dsDNA microarray. Fourthly, enzymatically extending hairpin primer to convert partial-dsDNA microarrays into complete-dsDNA microarray. The excellent efficiency and high accuracy of the enzymatic synthesis were demonstrated by incorporation of fluorescently labeled dUTPs in Klenow extension and digestion of dsDNA microarrays with restriction endonuclease. The accessibility and specificity of the DNA-binding proteins binding to dsDNA microarrays were verified by binding Cy3-labeled NF-kappaB to dsDNA microarrays. The dsDNA microarrays have great potential to provide a high-throughput platform for investigation of sequence-specific DNA/protein interactions involved in gene expression regulation, restriction and so on.     

Fabrication of high quality microarrays

Fabrication of DNA microarray demands that between ten (diagnostic microarrays) and many hundred thousands of probes (research or screening microarrays) are efficiently immobilised to a glass or plastic surface using a suitable chemistry. DNA microarray performance is measured by parameters like array geometry, spot density, spot characteristics (morphology, probe density and hybridised density), background, specificity and sensitivity. At least 13 factors affect these parameters and factors affecting fabrication of microarrays are used in this review to compare different fabrication methods (spotted microarrays and in situ synthesis of microarrays) and immobilisation chemistries.     

Sensitive detection of chromatin coassociations using enhanced chromosome conformation capture on chip

Chromosome conformation capture (3C) is a powerful technique for analyzing spatial chromatin organization in vivo. Technical variants of the assay ('4C') allow the systematic detection of genome-wide coassociations with bait sequences of interest, enabling the nuclear environments of specific genes to be probed. We describe enhanced 4C (e4C, enhanced chromosome conformation capture on chip), a technique incorporating additional enrichment steps for bait-specific sequences, and thus improving sensitivity in the detection of weaker, distal chromatin coassociations. In brief, e4C entails the fixation, restriction digestion and ligation steps of conventional 3C, with an optional chromatin immunoprecipitation (ChIP) step to select for subsets of chromatin coassociations, followed by bait enrichment by biotinylated primer extension and pull-down, adapter ligation and PCR amplification. Chromatin coassociations with the bait sequence can then be assessed by hybridizing e4C products to microarrays or sequencing. The e4C procedure takes approximately 1 week to go from tissue to DNA ready for microarray hybridization.     

Novel method for high throughput DNA methylation marker evaluation using PNA-probe library hybridization and MALDI-TOF detection

The methylation of CpG dinucleotides has become a topic of great interest in cancer research, and the methylation of promoter regions of several tumor suppressor genes has been identified as a marker of tumorigenesis. Evaluation of DNA methylation markers in tumor tissue requires hundreds of samples, which must be analyzed quantitatively due to the heterogeneous composition of biological material. Therefore novel, fast and inexpensive methods for high throughput analysis are needed. Here we introduce a new assay based on peptide nucleic acid (PNA)-library hybridization and subsequent MALDI-TOF analysis. This method is multiplexable, allows the use of standard 384 well automated pipetting, and is more specific and flexible than established methods, such as microarrays and MS-SNuPE. The approach was used to evaluate three candidate colon cancer methylation markers previously identified in a microarray study. The methylation of the genes Ade-nomatous polyposis coli (APC), glycogen synthase kinase-β-3 (GSK3β) and eyes absent 4 (EYA4) was analyzed in 12 colon cancer and 12 normal tissues. APC and EYA4 were confirmed as being differentially methylated in colon cancer patients whereas GSK3β did not show differential methylation.